Keratoconus (KC) is a noninflammatory disease characterized by thinning and scarring of the central cornea. We have been systematically investigating KC and have accumulated a significant body of information regarding the biologic defects associated with this disabling disease. We have shown that the levels of acid hydrolases, and cathepsins B and G are elevated in KC corneas as compared to normal human and other diseased corneas, whereas the levels of inhibitors such as alpha1-proteinase inhibitor (alpha1-PI) are diminished. These alterations are presumed to perturb the balanced degradation of corneal constituents. The aberrant degradative process is theorized to be the common pathway leading to KC. We have further found that transcription factor Sp1 (Specificity protein 1), at both mRNA and protein levels, is upregulated in KC corneas. During the past grant period, we demonstrated that Sp1 upregulation confers a downregulatory effect on the promoter activity of the a1-PI gene. Our hypothesis is that Sp1 upregulation is a key event that leads to many of the pathologic changes observed in KC. In this renewal application, we propose to investigate, in specific aim 1, the mechanisms by which Sp1 transcript and protein are upregulated in KC corneas. Standard nuclear run-on assays will be performed and the mRNA stability will be evaluated. Since the Sp1 protein is increased beyond the mRNA level, its turnover and phosphorylation and glycosylation status in KC corneas will also be examined and compared with controls. Experiments will in addition be conducted to determine whether the upregulated Sp1 mRNA level is related to aberrations in epigenetic regulatory mechanisms including DNA methylation and histone acetylation. Furthermore, Sp1 downregulation is observed during mouse corneal development. Mechanisms underlying such a downregulation will be explored. In specific aim 2, we will determine the in vivo effects of Sp1 overexpression in the cornea by targeting a Sp1 cDNA to corneal keratocytes under the control of keratocyte-specific keratocan promoter utilizing transgenic mouse technology. A binary mouse model will be used to engineer keratocyte-specific Sp1 expression under tetracycline control such that the phenotype can be switched on by feeding the animals doxycycline. Creation of the binary mouse line will be subcontracted to Dr. Chia-Yang Liu at the University of Cincinnati. The mouse will be examined to determine whether thinning of the corneas occurs, the time course, and the effects of environmental factors. In addition, apoptosis and oxidative damage have been proposed recently to be possible pathogenic mechanisms for KC and mutations in the transcription factor VSX1 gene have been identified in KC patients. In specific aim 3, we will investigate the roles of these factors in KC and their possible links to Sp1 overexpression. We will also complete the studies already underway to identify the genes differentially expressed in KC and their biological significance. Through these multiple approaches, we will further delineate cellular and molecular abnormalities in KC. We hope to gain a greater understanding toward the etiologic basis of KC and provide insights to ultimately lead to prevention and treatment of this corneal disease.